The biosynthesis of ACTH and endorphin will be studied in a mouse pituitary tumor cell line (AtT-20/D-16v) and in primary cultures of rat anterior and intermediate pituitary. Cells will be incubated with radioactive amino acids in both steady- and pulse-labeling experiments; immuno-chemical procedures utilizing antisera to ACTH and endorphin will be used to isolate endorphin-containing and ACTH-containing molecules from cell extracts and culture medium, and SDS polyacrylamide gel electrophoresis will be used to analyze the samples. In this way the biosynthetic pathways for ACTH and endorphin in these different tissues can be compared. Purification of mouse tumor cell betaLPH, 13K ACTH and 16K fragment by conventional protein purification procedures will be scaled up and contained. Purified molecules will be used for amino acid analysis, end-group determination, sequence determination, production of specific antisera and bioassay. The steroidogenic activity of the various forms of ACTH (and endorphin) purified from the mouse pituitary tumor cell culture medium will be assessed by using isolated rat adrenal cell cultures.